The overall goal of the proposed study is to investigate the role of cholesterol, including serum and islet cholesterol, in pancreatic (-cell dysfunction that characterizes the progression of type 2 diabetes. Alteration of pancreatic (-cell function leading to an impaired insulin secretory response to glucose is a hallmark of the transition from the prediabetic to the diabetic state. Among the many contributing factors, lipotoxicity, which refers to the diabetogenic effect of elevated circulating free fatty acids (FFAs) or cellular fat content, has been studied extensively. Despite the fact that cholesterol is often elevated in obese patients along with FFAs, the role of cholesterol in glucose-stimulated insulin secretion (GSIS) from pancreatic (-cells is not well documented. Unlike studies using mice fed on high-fat diets in which a rise in plasma cholesterol is accompanied by increased plasma FFAs, we established systems of isolated islets where cholesterol levels were altered, both in vivo using apoE-deficient mice and in vitro using pharmacological reagents, without changing cellular FFA levels. My preliminary data show a direct link between elevated serum cholesterol, elevated islet cholesterol and reduced GSIS, and that normal secretion can be restored by cholesterol depletion. I also show that the function of two proteins critically involved in GSIS, neuronal nitric oxide synthase (nNOS) and glucokinase (GK), depends on cholesterol-enriched membrane microdomains on the insulin granules. Through performing the proposed study I hope to solidify the conclusions from my preliminary data and provide a detailed molecular mechanism for cholesterol-dependent GSIS. The specific aims are: 1) Determine the role of cholesterol (dietary, plasma, and islet) in GSIS from pancreatic (-cells. 2) Identify the effect of cholesterol on GK regulation in pancreatic (-cells. 3) Investigate the role of membrane microdomains in nNOS regulation of GK in pancreatic (-cells. These studies will provide insights to a more complete understanding of obesity-induced type 2 diabetes and indicate the existence of a novel mechanism linking hyperlipidemia and pathogenesis of type 2 diabetes that is independent of FFAs. Relevance: (-cell dysfunction leading to decreased GSIS is considered the earliest and most relevant sign of type 2 diabetes mellitus. Studies on the link between hyperlipidemia and GSIS have focused mostly on FFAs. The direct effect of cholesterol on (-cell metabolism proposed in this study would open a novel set of mechanisms that may contribute to (-cell dysfunction and the onset of diabetes in obese patients.